Pancreatic islets isolated from β 2 adrenergic receptor knockout mice show reduced insulin secretion in response to nutrients

Activation of β2 adrenergic receptors by catecholamine or catecholamine-mimetic substances may enhance insulin secretion. We herein investigated KCland nutrient-stimulated insulin secretion in pancreatic islets isolated from β2 knockout (β2KO) mice. β2KO mice showed reduced body weight, fasting hypoglycaemia associate to a similar fasting insulinemia compared to control. β2KO mice also showed reduced glucose tolerance despite the higher sensitivity to insulin. Glucose-induced insulin secretion was impaired in pancreatic islets isolated from β2KO mice. Leucine-induced (20mM) insulin secretion was diminished in pancreatic islets isolated from β2KO mice when compared to control one. The depolarizing effect of KCl on insulin secretion was also impaired in pancreatic islets from β2KO mice. These results suggested a possible role of β2 adrenergic receptors on nutrient-induced insulin secretion.

Introduction D-glucose is the main physiological stimulus for insulin-secretion.After being transported into β-cells by GLUT-2, glucose is phosphorylated by a glucokinase and subsequently metabolized through the glycolytic pathway.When glucose concentration is elevated (above 7 mM), the ATP/ADP ratio also increases, leading to the closure of the ATP-sensitive K + channel (K ATP channel).This event ends up depolarizing the plasma membrane and opening the voltage-sensitive Ca 2+ channels, which in turn increases the intracellular concentration of calcium and causes the exocytosis of insulin granules into the bloodstream (MACDONALD et al., 2005).Amino acids (AA) such as leucine and arginine are insulin secretion inducers when the serum glucose concentration is high.AA depolarize the plasma membrane (by closing the K ATP channel) and increase calcium content in pancreatic beta cells (HENQUIN et al., 2003).
Mice with total deletion of beta adrenoceptors showed glucose intolerance with increased sensitivity to insulin, indicating a possible alteration in insulin secretion (ASENSIO et al., 2005).However, the subtype of adrenergic receptor involved is still unknown.In this regard, we used β 2 KO mice to investigate the impact of β 2 adrenergic receptor on whole-body glucose homeostasis and insulin secretion on pancreatic islets.
Pancreatic islets from β 2 KO mice displayed reduced insulin secretion when incubated with glucose, amino acids or KCl.These results suggest that the β 2 adrenergic receptors play a key role in the nutrientinduced insulin secretion.

Animals
Heterozygote β 2 -AR +/-mice were mated to generate both homozygote β 2 -AR +/+ and -/mice.Several matings were carried out to obtain knockout mice for the β 2 -AR -/-(β 2 KO) (CHRUSCINSKI et al., 1999;MEDEIROS et al., 2008).Four-month-old mice were used.Each group of five animals was kept in cages under controlled temperature (23 ± 2ºC) and light (12h light/dark cycle) conditions.The animals were given a commercial chow (Nuvital, Brazil) and water ad libitum.All experimental protocols were approved by the Ethics Committee on Animal Experimentation of the Institute of Biomedical Sciences of the University of São Paulo, São Paulo State, Brazil.

Chemicals
Rat insulin labeled with 125 I was obtained from Amershan Pharmacia (São Paulo, SP, Brazil).All other reagents were obtained from Sigma, unless otherwise mentioned.Insulin antibody was a gift from Dr. Leclercq-Meyer, Université Libre de Bruxelles, Belgium.

Insulin tolerance test (iTT) and the percentage of periepididymal fat pad
After a 4-hour fast, animals were anesthetized with thiopental (40 mg kg -1 body weight).Insulin diluted in saline solution (0.9%) was then immediately intraperitoneally injected (25 mU g -1 body weight) and blood samples were collected from the tail at 0, 5,10,15,20,25,30,40,60 and 120 min.after injection for glycaemia determination.Subsequently, these same mice were decapitated, and then, abdominal cavity of animals from both groups were open, periepididymal fat pads were collected and weighed to determine epididymal fat pad content (% periepididymal fat pad = g g -1 body weight × 100).

Isolation of the pancreatic islets
Pancreatic islets were obtained from mice as previously described (LATORRACA et al., 1999) with few modifications.The pancreas was inflated with Hanks solution containing type V collagenase (35 mg mL -1 ), kept at 37ºC for 20 min. in a water bath and stirred for an additional 1 min.They were then washed with a Krebs-Henseleit buffer solution containing 115 mM NaCl, 5 mM KCl, 24 mM NaHCO 3 , 1 mM CaCl 2 and 1 mM KCl 2 .The islets were then collected using a magnifying glass.

Incubation of the pancreatic islets
A group of five islets was transferred to plates containing 1 mL Krebs-Henseleit buffer solution supplemented with 0.125% albumin for 60 minutes at 37ºC in the presence of glucose (5.6 mM).This solution was bubbled with a mixture of O 2 (95%) and CO 2 (5%).After the pre-incubation period, the incubation with different glucose concentrations (2.8, 5.6, 8.3, 11.1 and 16.7 mM) took place for an additional 60 min.Another group of islets was homogenized and the total insulin content was determined by radioimmunoassay (RIA).For the tests carried out with amino acids, pancreatic islets were incubated with Lleucine (20 mM) and/or L-arginine (20 mM) with or without glucose (8.3 mM), as previously described (LATORRACA et al., 1999).Furthermore, incubation with a high concentration of KCl (30 mM) was carried out in the presence of glucose (8.3 mM).At the end of incubation, insulin was measured by RIA.Aliquots were collected from the incubated pancreatic islets and plasma insulin concentrations were measured by RIA using rat insulin marked with 125 I from Amershan Pharmacia (São Paulo, São Paulo, Brazil).

Statistical analysis
The results obtained with both mice strains were presented as mean ± SEM.The data were analyzed using Student's t-test for two conditions and a two-way ANOVA test with Bonferroni post hoc test for more than two conditions, and then systematized by GraphPad Prism, 4.00 for Windows (GRAPHPAD PRISM, 2003).

Results and discussion
Body weight, epididymal fat pad content, fasting blood glucose and insulin levels of 4-month-old β 2 KO and wild-type mice were measured.β 2 KO mice presented both reduced body weight and perigonadal fat compared to wild-type mice (Table 1).Fasting blood glucose levels were 26% lower in β 2 KO animals (p < 0.05).No difference was detected between the groups for plasma insulin levels.However, HOMA was enhanced in β 2 KO mice when compared to controls (Table 1).The results are representative of 5 distinct experiments and are described as mean +/-SEM.The asterisk, *, indicates p < 0.05.The number of animals is indicated in brackets.
Glucose overload in the gTT causes a more pronounced increase in blood glucose levels in β 2 KO mice at 15 (125%) and 20 (134%) min compared to control (Figure 1A).As expected, insulin infusion induced a reduction in blood glucose levels in both wild-type and knockout mice.However, the magnitude of this effect was greater in β 2 KO mice at the 15, 20 and 25 min time points after insulin injection by 20, 36, and 33%, p < 0.01, respectively (Figure 1B).AUC-glucose was significantly diminished in the β 2 KO mice compared to control, p < 0.01 (Figure C insert at Figure 1B).These results were corroborated by the HOMA test (Table 1).
Reduced insulin secretion in response to glucose stimulus was observed in pancreatic isolated islets from β 2 KO mice, as indicated by the increased EC 50 of glucose stimulus compared to wild-type mice; EC 50 of 11.4 and 8.1 mM, p < 0.05 (Figure 2).8, 5.6, 8.3, 11.1 and 16.7 mM glucose for 60 min.The data are expressed as means ± SEM by total insulin content (ng islets -1 ) of 7 distinct experiments with triplicate.A two-way ANOVA test followed by a Bonferroni test was run.*p < 0.05.
Although leucine is able to enhance glucoseinduced insulin secretion, this effect was abolished in pancreatic islets isolated from the β 2 KO mice (Figure 3), however, this response was not observed in pancreatic islets incubated only with arginine (Figure 3).), Leu (20 mmol L -1 ), Arg (20 mmol L -1 ), Gluc+Leu and Gluc+Arg, KCl (30 mM), same concentrations as for the individual stimuli.The data are expressed as means ± SEM by total insulin content (ng islets -1 ) of 7 experiments with triplicate.*p < 0.05.
The insulin secretion induced by the hyperpolarizing agent KCl was also reduced to 43% of that detected in the isolated pancreatic islets of wild-type (Figure 3).Furthermore, the total insulin content was reduced in pancreatic islets β 2 KO mice as compared to the control group (WT = 125.2± 5.6 vs β 2 KO = 102.8± 8.5 ng islets -1 , p < 0.05) (Figure 4).β 2 KO mice showed lower body weight, as reported by Chruscinski et al. (1999).Those animals are leaner when compared to the control group, and that result was evidenced by the reduced weight of the periepididymal fat pad.The amount of perigonadal fat is considered a predictor of obesity in rodents according to Rogers and Webb (1980).
β-adrenergic stimulation inhibits insulinstimulated glucose uptake in skeletal muscle and in white adipose tissue.The epinephrine decreases insulin-stimulated glucose transport in skeletal muscle from both rats and humans, and in rat adipose cells through reduction of GLUT4 translocation to the plasma membrane as demonstrated by Han and Bonen (1998), Bonen et al. (1992), Jones and Dohm (1997), Laurent et al. (1998), Nishimura et al. (1991), and Yang et al. (2002).However, White andKahn (1994), andSaltiel andKahn (2001) showed that insulin is the most important extracellular signaling agent that leads to glucose uptake.Alterations in the intracellular insulin signaling are associated with the development of insulin resistance and DM2 according to Carvalho et al. (1999), Marçal et al., (2012), and Yamauchi et al. (1996).Phosphorylation of both insulin receptor and insulin receptor substrates, IRS-1 and IRS-2 is reduced when the beta adrenergic receptor is activated as well demonstrated by Doronin et al. (2002).In this regard, the improvement in insulin sensitivity reported herein in β 2 KO mice is in agreement with the established effects of catecholamines on intermediary metabolism.Miller (1981) described that pancreatic islets have an abundant sympathetic innervation.According to this author, Peterhoff et al. (2003) described that the α 2 -adrenergic receptors when activated by epinephrine or adrenomimetics, causes inhibition of insulin secretion.On the other hand, β 2 -adrenergic receptors have a dual effect, stimulating or inhibiting insulin secretion as well as pointed out by Narimiya et al. (1981), Marçal et al. (2006), Ahrén andLundquist (1981), andKurose et al. (1990).The reduced glucosestimulated insulin secretion (GSIS) observed in β 2 KO mice could be associated with a prolonged activation of α-adrenergic receptors as demonstrated by Sjoholm (1991).Another interesting observation was the reduced effect of glucose plus arginine and leucine on insulin secretion.Despite the fact that the mechanism of L-arginine-induced insulin secretion is not fully clarified, many authors suggest that the potentiation of glucose-induced insulin secretion by L-arginine is mediated by β-cell membrane depolarization due to the electrogenic influx of the cationic amino acid into the β-cell as described by Charles et al. (1982), Henquin and Meissner (1981), Blachier et al. (1989), Hermans et al. (1987), and Smith et al. (1997).Thus, amino acid depolarizes the plasma membrane, whose effect is enhanced by glucose as shown by Hermans et al. (1987), and stimulates Ca 2+ influx by activation of voltage-sensitive Ca 2+ channels according to Hermans et al. (1987), Smith et al. (1997), and Weinhaus et al. (1997).
According to the findings from Sener and Malaisse, (1980), Gylfe (1976), Panten et al. (1972), and Carpinelli and Malaisse (1981), L-Leucine induces insulin secretion by two mechanisms: (i) enhanced mitochondrial metabolic activity through activation of WT ß 2 KO glutamate dehydrogenase, and (ii) by transamination to α-ketoisocaproate and subsequent entry into the TCA cycle via acetyl-CoA leading to an increase in ATP production.Alteration of insulin secretion in pancreatic islets from β 2 KO mice could be related to a reduction in the uptake and/or metabolism of these nutrients.
The electrical activity of beta cell membrane, through Ca 2+ -dependent action potentials, plays a key role for insulin secretion: the secretion of insulin is abolished in the absence of calcium in agreement with MacDonald et al. (2005), Henquin et al. (2003), Carpinelli andMalaisse (1981), andAshcroft (2005).However, change in function and/or expression of voltage-gated Ca 2+ channels cannot be ruled out in isolated pancreatic islets from β 2 KO mice.

Conclusion
We have demonstrated herein that β 2 -adrenoceptor deletion induces remarkable modifications of glucosestimulated insulin secretion in isolated pancreatic islets.The depolarization of beta cells plasma membrane is involved in the effect and deserves to be further investigated.

Figure 1 .Figure 2 .
Figure 1.β 2 KO mice had altered glucose tolerance test (A) with increased insulin sensitivity (B).The data are expressed as percentage of the basal blood glucose level (initial time (t = 0)) of each β 2 KO (open circle) and wild-type (black square) mice.The AUC-glucose represents the values obtained during insulin sensitivity test (insert on Figure1B).The analysis was performed with 4 animals of each strain.A two-way ANOVA test followed by a Bonferroni test was run.*p < 0.01.

Figure 4 .
Figure 4. Insulin content in isolated pancreatic islets from wild-type (dark bar) and β 2 KO (open bar) mice.The data are expressed as means ± SEM of 7 mice of each strain with triplicate.*p < 0.05.

Table 1 .
Body weight, visceral adipose tissue, blood glucose level and serum insulin level of wild-type and β 2 KO mice.